Molecular dissection of Ca2+ efflux in immortalized proximal tubule cells

Kenneth E. White, Frank A. Gesek, Teresa Nesbitt, Marc K. Drezner, Peter A. Friedman

Research output: Contribution to journalArticle

18 Scopus citations

Abstract

Plasma membrane Ca2+-ATPase (PMCA) and the Na+/Ca2+ exchanger participate in regulating cell function by maintaining proper intracellular Ca2+ concentrations ([Ca2+](i)). In renal epithelial cells these proteins have been additionally implicated in cellular calcium absorption. The purpose of the present studies was to determine the Ca2+ extrusion mechanisms in cells derived from the proximal tubule. Homology-based RT-PCR was used to amplify PMCA transcripts from RNA isolated from mouse cell lines originating from the S1, S2, and S3 proximal tubule segments. S1, S2, and S3 cells exhibited only PMCA1 and PMCA4 products. PCR product identity was confirmed by sequence analysis. Northern analysis of proximal tubule cell RNAs revealed appropriate transcripts of 7.5 and 5.5 kb for PMCA1 and 8.5 and 7.5 kb for PMCA4, but were negative for PMCA2 and PMCA3. Western analysis with a monoclonal antibody to PMCA showed that all proximal cell lines expressed a reacting plasma membrane protein of 140 kD, the reported PMCA molecular mass. Na+/Ca2+ exchanger (NCX1) mRNA expression, analyzed by RT-PCR, protein expression by Western analysis, and functional exchange activity were uniformly absent from all proximal tubule cell lines. These observations support the idea that immortalized cells derived from the proximal tubule express PMCA1 and PMCA4, which may serve as the primary mechanism of cellular Ca2+ efflux.

Original languageEnglish (US)
Pages (from-to)217-228
Number of pages12
JournalJournal of General Physiology
Volume109
Issue number2
DOIs
StatePublished - Feb 1997

Keywords

  • Ca-ATPase
  • Na/Ca exchange
  • PMCA
  • calcium transport
  • kidney

ASJC Scopus subject areas

  • Physiology

Fingerprint Dive into the research topics of 'Molecular dissection of Ca<sup>2+</sup> efflux in immortalized proximal tubule cells'. Together they form a unique fingerprint.

Cite this